Interpretive Summary: In 1996, approximately one billion gallons of fuel ethanol were produced from corn starch. Demand for ethanol, as a substitute for petroleum, is expected to increase because of concerns related to national security, economic stability, environmental impact, and global warming. Expanding fuel ethanol production will require developing alternative feedstocks. In order to expand fuel ethanol production, we are exploring the fermentation of the sugars derived from corn fiber and agricultural wastes plus developing new ethanol-producing microorganisms to use these materials. We have recently developed a novel bacterial strain suitable for repeated batch or continuous fermentation of mixed sugars. The strain is superior to previous bacterial strains developed for ethanol fermentation because it is genetically more stable. The strain is able to ferment the sugars found in corn fiber into ethanol with 91-92% of the maximum possible yield.

Technical Abstract:
We have developed a novel ethanologenic Escherichia coli strain (FBR3) suitable for continuous fermentation of feedstocks containing mixed sugars, such as lignocellulosic hydrolysates, into ethanol. This strain carries the pLOI297 plasmid, which contains the genes from Zymomonas mobilis necessary for efficiently converting pyruvate into ethanol. Strain FBR3 selectively maintains pLOI297 when grown anaerobically. Cultures of strain FBR3 were serially transferred 10 times in aerobic and anaerobic cultures with sugar limited medium containing either glucose or xylose, but no selective antibiotic. An average of 97.4 of the cells maintained pLOI297 in anaerobic cultures. In contrast, the plasmid quickly disappeared from aerobic cultures. Plasmid maintenance depends upon deletion of two enzyme activities: pyruvate formate lyase (pfl) and lactate dehydrogenase (ldh). The later mutation was introduced by chemical mutagenesis. The stability of this mutation was confirmed by the absence of hydrogen gas production, an indirect assay for pfl activity, in each of the cultures. The FBR3 culture transferred on xylose containing medium was tested in pH controlled batch fermentations to determine if the strain still efficiently converted pentoses and hexoses into ethanol. Batch fermentation medium contained either 10% w/v arabinose, glucose, xylose, or a mixture of these sugars. Fermentations were completed in 70-80 h and ethanol yields were 90-91% of theoretical; maximum ethanol concentrations were 4.38-4.66% w/v.